Polymerase chain reaction (PCR) technology can be used to detect the presence of small amounts of particular target polynucleotide sequences through the well known cyclic amplification scheme. The initial quantity of the target nucleic acid can be determined by detecting the amount of amplification product (amplicons) after a certain number of amplification cycles, or using real time PCR techniques that monitor signals from amplicons as they are generated. However, the linearity of PCR amplifications becomes poor with extended cycles. Further, typical real time PCR assays are based on the quantification cycle (CQ) the fractional cycle number where fluorescence increases above the threshold, inherently limited by high background and large increments between reportable values.
For example, real time PCR can be carried out as follows. The process starts with provision of PCR reaction constituents, e.g., at least one pair of specific primers, deoxyribonucleotides, a suitable buffer solution, and a thermo stable DNA polymerase are added to a sample expected to contain a target polynucleotide of interest. If the target sequence is present, anti-sense copies of the sequence between the primer pair will be synthesized by the polymerase enzymes. The product can be melted and the synthesis repeated to make copies of the newly synthesized anti-sense nucleic acids. Further repetitions of polymerization and melting can geometrically expand the number of copies from the original target polynucleotide. A substance marked with a fluorophore can be added to the PCR reaction mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore after it has been excited at the required wavelength allowing the generation rate of new target polynucleotide copies to be detected as ever increasing fluorescent signals. See, e.g., Dual Resonance Energy Transfer Nucleic Acid Probes, U.S. Pat. No. 7,081,336, by Bao, et al. These measurements can be made after each amplification cycle to generate a trend chart with parameters roughly related (e.g., by a factor of 2, at best) to the initial target sequence quantity. Even these poor results require initial extraction of nucleic acids before analysis can begin.
Because the primer probes can hybridize to imperfect targets, the standard PCR and quantitative PCR methods can have problems accurately detecting target sequences with only minor variants. One way this problem has been addressed is by using peptide nucleic acid (PNA) clamping probe schemes. For example, in Methods and Kits for the Detection of Nucleotide Mutations Using Peptide Nucleic Acid as Both PCR Clamp and Sensor Probe, U.S. Pat. No. 7,803,543, by Chiou, et al., PNA clamping probes to wild type target block amplification of the target nucleic acid unless there is a mutation in the PNA probe footprint. In Chiou, confirmation of sequence variants depends on comparison of PNA and PCR product melting temperatures. However, this and related techniques suffer from ungainly detection procedures, and are not well adapted to precise and accurate quantitative measurements.
There remains a need for sensitive and quantitative methods to detect target nucleic acids in samples. Significant benefits would be provided by solutions to the problem of detecting single base mutations in target nucleic acid sequences of interest. It would be a substantial advancement if rare DNA sequence quantitation could be practiced on crude lysate samples. The present invention provides these and other features, which will be apparent upon complete review of the following.